The homeobox is a nuclear DNA-binding protein domain, discovered in genes controlling cell fate commitment and differentiation in Drosophila melanogaster. The domain is present in murine and human genes with very specific temporal and spatial expression patterns during embryogenesis. It is also part of specific transcription factors for human effector genes.Complex patterns of lineage-restricted expression of homeobox genes in hematopoietic cell lines have been observed. This proposal seeks to elucidate the role of these genes in cell commitment and maturation in hematopoiesis. A sensitive screening system based on the polymerase chain amplification was established to readily distinguish multiple homeobox gene transcripts in hematopoietic progenitor lines. The system allows a first rapid targeting of lineage- and maturation-specific homeobox transcripts. If restriction of expression according to lineage and maturation stage is confirmed by hybridization analysis with full length cDNA probes, functional relevance will be established by two modes of investigation: Loss of function by mRNA ablation in cell lines with antisense techniques and gain of function by forced expression in non-expressing cell lines. If the master gene function is clearly shown with this assay, we will search for lesions in the gene in primary leukemic cells and a dissection of the sectional components of the gene: cis-acting sequences and coding domains. Antisera will be used to look at post-translational events where there is very strong suspicion for a functional role as phosphorylation and protein complex formation. Studies will be extended to the whole animal with transgenic constructs and chimeric mice where the homeobox gene is disrupted by homologous recombination. This research will provide strong and broad background in developmental and molecular genetics and in the cell biology of differentiation.